It has long been thought that long-chain free fatty acids (FFAs) stimulate insulin secretion via mechanisms involving their metabolism in pancreatic β-cells. Recently, it was reported that FFAs function as endogenous ligands for GPR40, a G protein-coupled receptor, to amplify glucose-stimulated insulin secretion in an insulinoma cell line and rat islets. However, signal transduction mechanisms for GPR40 in β-cells are little known. The present study was aimed at elucidating GPR40-linked Ca²⁺ signaling mechanisms in rat pancreatic β-cells. We employed oleic acid (OA), an FFA that has a high affinity for the rat GPR40, and examined its effect on cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in single β-cells by fura 2 fluorescence imaging. OA at 1–10 μM concentration-dependently increased [Ca²⁺]_i in the presence of 5.6, 8.3, and 11.2 mM, but not 2.8 mM, glucose. OA-induced [Ca²⁺]_i increases at 11.2 mM glucose were inhibited in β-cells transfected with small interfering RNA targeted to rat GPR40 mRNA. OA-induced [Ca²⁺]_i increases were also inhibited by phospholipase C (PLC) inhibitors, U73122 and neomycin, Ca²⁺-free conditions, and an L-type Ca²⁺ channel blocker, nitrendipine. Furthermore, OA increased insulin release from isolated islets at 8.3 mM glucose, and it was markedly attenuated by PLC and L-type Ca²⁺ channel inhibitors. These results demonstrate that OA interacts with GPR40 to increase [Ca²⁺]_i via PLC- and L-type Ca²⁺ channel-mediated pathway in rat islet β-cells, which may be link to insulin release.